1. Field of the Invention
The present invention relates to isolating pure plasmid DNA from E. coli and other organisms. The addition of indicator dyes to the alkaline lysis based buffers allows for easy and rapid visual monitoring of the resuspension, lysis, and neutralization steps. The ability to assess the process increases plasmid yield and also reduces errors. The present invention is useful in other nucleic acid purification applications.
2. Description of the Related Art
The present invention relates to the purification of plasmid DNA and other nucleic acids from source materials. The purification of plasmid DNA from bacterial lysates is a standard molecular biological technique that is critical for downstream recombinant DNA manipulations. It is critical and consumes significant resources of academicians and scientists in biotechnology research and development. The purity and quantity of the plasmid DNA are important factors that remain troublesome for the entire scientific community. The sensitive reactions commonly employed in molecular biology experiments of reverse transcription, transcription, DNA and RNA sequencing, polymerase chain reaction (PCR), restriction digests, ligation reactions, end modifications, among other similar based modification procedures require the plasmid DNA, or other nucleic acid molecules be free from contaminants. It is also desirable to isolate the nucleic acid in significant quantities to ensure a reliable source of material with which to proceed to additional experiments. In many instances there is a need to move a desired plasmid DNA, or fragment thereof, through several manipulations to reach the desired endpoint. Cloning procedures are often complex and involve numerous steps and so methods that reliably isolate pure plasmid DNA, and other nucleic acids, in significant quantities are desired. Contaminants are and remain a persistent problem and the majority of them often result from the initial steps of lysis of the bacteria through clearing of the lysate. In addition, the lysis and neutralization, or clearing steps, are often the points where significant loss of the desired plasmid DNA, or nucleic acid, is encountered.
Conventional procedures for isolating plasmid DNA includes harvesting the bacterial cells and obtaining the plasmid DNA, or other target nucleic acid, in a pure form via lysis, free from the undesirable contaminating medium and the cellular constituents. This is typically called a cleared bacterial or cellular lysate. The cell lysis may be performed in a variety of ways including mechanical sonication or blending, enzymatic digestion and also the traditional chemical means of alkaline lysis. The alkaline lysis based protocols remain the basis for many plasmid purification methods, though other procedures such as the boiling lysis, triton lysis, polyethylene glycol protocols are also used (Bimboim and Dolly, 1979 Nucl. Acids Res. 7:1513-1523; H. Bimboim, 1983, Meth. in Enzym., vol. 100, pp. 243-255, Holmes and Quigley, 1981, Anal. Biochem. 114:193-197; Clewell and Helinski, 1970, Biochemistry, 9:4428-4440; Lis and Schleif, 1975 Nucl. Acids Res. 2:757). The dominant alkaline lysis method's lysis and neutralization steps are important points where loss is observed and cellular contaminants are introduced, often unnecessarily.
Approaches that coupled alkaline lysis to cesium chloride gradient centrifugation and organic extraction with toxic and caustic phenol/chloroform and alcohols have largely been replaced by a variety of systems that utilize coupling a cleared lysate to rapid and efficient chromatographic methods. The observation that DNA bound preferentially to ground glass or glass fiber disks in the presence of high concentrations of sodium iodide or sodium perchlorate has allowed the development of new purification methodology based on these results (Marko et al. 1981, Analyt. Biochem. 121:382-387, Vogelstein et al. 1979, Proc. Nat. Acad. Sci. 76:615-619). The use of the chaotropic salt solutions such as guanidinium, iodide, perchlortate, and trichloroacetate coupled to forms of silica based or other chromatographic techniques, has resulted in a preferred methodology for plasmid as well as general nucleic acid purification.
These systems often employ the use of such chaotropic salts together with chromatographic techniques either in small or large scale formats that are typically based on the silica based materials of diatomaceous earth, silica particles, silica resins, silica embedded filters, magnetic silica particles, and different combinations thereof. In addition modified silica materials and ion exchange resins are also commonly used, either alone or in combinations in the form of hybrid resins. Additionally other chromatographic techniques have been commonly used in nucleic acid purification procedures. Despite these improvements and the development of numerous systems based on these technologies there remains a need to develop improved systems to satisfy demands for easier, faster protocols with increased yield and reliability for high-level quantity purification of plasmids and other nucleic acid materials.
Generally, the plasmid or nucleic acid is bound to the matrix using vacuum filtration or centrifugation methods, washed on the column similarly and finally eluted with water, TE buffer, or other elution buffer. Many preparations have been developed commercially, such as the Wizard.™ DNA purification line of products (Promega Corporation); or the Qiagen line of DNA isolation systems from. (Qiagen Corp.), the FlexiPrep (Pharmiacia), and GeniePrep (Ambion), among others.
All of these commercial procedures do not adequately address the formation of the cleared lysate from which the plasmid, or nucleic acid, is to be purified. The commercial market for plasmid purification has developed to a multimillion dollar industry, but has focused largely on the final chromatographic purification steps. Thus most of the advances in plasmid purification ignore the early steps of nucleic acid isolation that are crucial for the yield and quality of the isolated product.
The means of preparing a cleared bacterial, or other biological source of lysate, is a point that has remained problematic and overlooked. Ensuring that complete lysis and neutralization occurs can be more important for yield and also for eliminating contaminants, than the later chromatographic steps. Quite often inefficient lysis and neutralization leads to contaminants being carried over into later steps and dramatic reduction in yield. Advances in these early steps in plasmid and nucleic acid purification procedures will ensure that the significant improvements and modifications in silica matrixes, particles, and chromatographic procedures, have cleaner and more abundant starting material. Other problems associated with many current techniques are inefficient sample handling, overall time delay, and reduced yields. Improved visual recognition of lysis would improve overall quality and yield, especially when the nucleic acid in question is in low supply due to intrinsic factors such as low copy number, toxicity, or limited availability of the starting biological material.